Duncan Mark T, Shin Seungjin, Wu Jia J, Mays Zachary, Weng Stanley, Bagheri Neda, Miller William M, Shea Lonnie D
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208.
Biotechnol Bioeng. 2014 Oct;111(10):2082-94. doi: 10.1002/bit.25262. Epub 2014 Jul 14.
The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation.
巨核细胞 - 红系祖细胞（MEP）向红系（E）或巨核细胞系（MK）谱系的定向分化可增强红细胞和血小板的体外生成，用于治疗性输血。MEP分支处的谱系选择在很大程度上受细胞内信号转导网络活性的控制，其输出决定转录因子（TFs）的活性并最终决定基因表达。尽管许多TFs与之相关，但E或MK分化是一个需要数天的复杂过程，而在定向和终末成熟过程中TF活性的动态变化相对未被探索。在此，我们应用活细胞阵列对MEP分支过程中TF活性进行大规模、动态定量分析。在双能K562细胞向E和MK分化过程中，对一组造血TFs（GATA - 1、GATA - 2、SCL/TAL1、FLI - 1、NF - E2、PU.1、c - Myb）进行了表征。确定了与向每个谱系分化相关的动态TF活性谱，并与先前的报道进行了验证。从这些活性谱中，我们表明GATA - 1在早期血红素和佛波酯诱导的分化过程中是一个重要枢纽，并揭示了E和MK分化的几个特征性TF相互作用，证实了文献中记载的调控机制。此外，我们突出了E和MK分化不同阶段的几个新型TF相互作用。此外，我们使用MK定向细胞系CHRF - 288 - 11（CHRF）研究了烟酰胺（NIC）促进终末MK成熟的机制。与其对多倍体的增强作用相伴，NIC强烈增强了已知参与终末MK成熟的三个TFs的活性：FLI - 1、NF - E2和p53。TF活性的动态分析代表了一种新颖的工具，可补充侧重于mRNA和蛋白质表达水平的传统检测方法，以了解祖细胞分化。
